A. Field of the Invention
The present invention relates generally to the fields of molecular biology, gene regulation and pathology. More specifically, in certain aspects, the invention relates to the identification of premalignant or malignant conditions in tissues. In other aspects, the invention relates to methods and compositions for the identification of apoptosis, or programmed death, in cells. In particular, the present invention relates to monitoring of levels of apurinic/apyrimidinic endonucleases, also known as APE""s.
B. Related Art
(i) Cancer Markers
Despite continued efforts worldwide to identify useful prognostic factors for premalignant and malignant conditions (hereinafter together referred to as xe2x80x9c(pre)malignant conditionsxe2x80x9d) of human tissues, relatively few markers and associated screens have been discovered which reliably identify (pre)malignant conditions. As one specific example, squamous cell carcinoma of the cervix uteri (SCCC) is the second most common female malignancy, and the leading cause of death by cancer in women worldwide (Mitchell et al., 1995; Burger et al., 1995; Richard et al., 1995). Based on recent estimates, there will be approximately 15,800 new cases of invasive disease, 65,000 cases of carcinoma in situ (CIS; premalignant), and 4800 deaths attributed to SCCC annually in the United States alone (American Cancer Society, 1995). African-and Hispanic-American women and poor Caucasian women were found to have a mortality rate from cervical cancer of more than double that of all Caucasian-American women (Burger et al., 1993; Miller et al., 1993; Davis et al., 1995; Parham et al. 1995), and HIV positive women were diagnosed with CIS at five times the rate of HIV negative women (Wright et al., 1994; Heard et al., 1995).
Cervical cancer arises in the squamous cells lining the cervix tissue. Precancerous lesions are known as CIS, dysplasia, or cervical intraepithelial neoplasia (CIN). Although the development of these cells into invasive carcinoma can take ten to twelve years, in about 10% of patients the development is much more rapid, occurring in less than a year (National Cancer Institute, 1995). Early detection of cervical cancer substantially increases the probability of survival, with both malignant and premalignant conditions being detectable by the so-called Pap smear.
While the Pap smear is relatively widely used, and has had an overall positive impact on women""s health, it presents several significant drawbacks. Pap smear sampling must be performed by highly trained clinicians to result in an interpretable, representative sample of the cells lining the cervix (Koss, 1989). As well, a trained cytologist must analyze the morphology of the cells upon microscopic examination (Koss, 1989). Significant human error is attributed to both steps, contributing to high levels of false-negative readings (Koss, 1989; Koss, 1993). A majority of studies estimate the rate of false-negatives at 20%-30%, with various other studies putting this value at 5% to in excess of 50% (Morell et al., 1982). In addition, subsequent to a positive interpretation of a Pap smear, a physician typically biopsies the cervical tissue to confirm the diagnosis and assist in the determination of the stage of the disease and the design of an appropriate treatment regimen. The biopsy sample is analyzed by a pathologist for the presence or absence of (pre)malignant cells and to determine the extent of tumor growth. Human error can also arise in these procedures (Sideri, et al., 1982).
In light of these and other shortcomings of the common Pap smear, researchers have been actively seeking a reliable marker for (pre)malignant states in cervical tissue. A useful marker and associated assay are understood to require a number of attributes. An assay using the marker must consistently detect differences in cancer and noncancer, and exhibit both specificity (few false positives) and sensitivity (few false negatives). Quantitative assays find increased utility over those which are merely qualitative, and cancer marker specific for a particular organ or cell type will be more useful for initial screening purposes, but organ/cell specificity is less important for monitoring previously diagnosed patients.
A number of putative markers for (pre)malignant conditions of the cervix have been identified; however, the markers suggested to date exhibit several shortcomings. For instance, squamous cell carcinoma antigen is a glycoprotein purified from SCCC that has been found to be a marker for cancerous conditions of the cervix (Kato et al., 1982; Kato et al., 1984). This marker was originally called T-4 in a lesser-purified form, and serum SCCA was found to be elevated in 61% of SCCC cases overall, ranging from 30-45% in Stage 1 to 90-100% in Stage 4 (Crombach et al., 1989) (FIGO classification, National Cancer Institute, 1995). In original testing of SCCC as a tissue marker using flow cytometry of vaginal smear cells, 85% of SCCC cases, 80% of severe and 43% of mild to moderate dysplasias and 21% of normal specimens contained cells stained with antibodies to SCCA (Suehiro et al., 1986). This lack of specificity decreases the usefulness of SCCA as a marker, which has also been bolstered by the observation that cytosolic concentration of SCCA in normal cells is twice as high in normal cells than in SCCC cells (Crombach et al., 1989).
Another putative marker for SCCC is carcinoembryonic antigen (CEA). One of the most studied antigens using immunohisto hemical analysis for the determination of neoplastic cells in SCCC is CEA. Reports as to the percentage of different dysplastic and neoplastic lesions stained have varied (Toki et al., 1991; Rutenan et al., 1978; van Nagell et al., 1982; Bamford et al., 1983; Bychkov et al., 1983; McDiken et al., 1983; Lindgren et al., 1986; Agarwal et al., 1990). Additional possible markers which have been studied, with varying degrees of success, include proliferating cell nuclear antigen (PCNA) (Raju, 1994; Steinbeck et al., 1995), epithelial membrane antigen (EMA) (Bamford et al., 1983; Sarker et al., 1994), various keratins (Rajur et al., 1988; Auger et al., 1990), Tn antigen (Hamada et al., 1993; Hirao et al., 1993), oncogenes and tumor suppressor genes (Kohler et al., 1989; Tervahauta et al., 1994; Hale et al., 1993; Terzano et al., 1993; Sainz et al., 1993; Tervahauta et al., 1993; Cardillo et al., 1993), and various others (Fuchs et al., 1989; Flint et al., 1988; Costa et al., 1987; Lara et al., 1994; Carico et al., 1993; Harlozinski et al., 1985).
(ii) APE
Apurinic/apyrimidinic endonucleases (hereinafter sometimes referred to as xe2x80x9capurinic endonucleasexe2x80x9d or xe2x80x9cAPExe2x80x9d) catalyze repair of baseless sites in DNA. At least 10,000-20,000 of these sites are generated daily in every human cell as a result of oxidation, spontaneous hydrolysis, and the removal of modified bases by DNA glycosylases (Loeb, 1985, FIG. 15). These baseless sites disrupt transcription and are highly mutagenic if not repaired.
The major human apurinic/apyrimidinic endonuclease is a 37,000 Dalton protein which has been cloned and shown to complement APE deficient bacteria (Demple et al., 1991). APE has been shown to be identical to Ref-1, a redox factor facilitating the DNA binding of a number of transcription factors, many of which are important in oncogenesis, including Fos, Jun, Myb, and members of the ATF/CREB family (Xanthoudakis et al., 1992). Recently, APE has also been shown to be involved in the negative regulation of transcription of the parathyroid hormone gene by extracellular calcium in vitro (Okazaki et al., 1994). Ape also appears to be a major regulator of p53 activity, acting through protein modification of p53 (Jayaraman et al., 1997).
Besides DNA repair activity, the major human APE repair enzyme has been found to exhibit multiple functions, many by in vitro studies. For example, investigators studying Ref-1, a redox regulating transcription factor, discovered that Ref-1 and APE were identical (Xanthoudakis et al., 1992). APE/Ref-1 facilitates the DNA binding characteristics of Jun-Jun homodimers, Fos-Jun heterodimers, HeLa AP-1, and numerous other transcription factors, including Myb, members of the CREB family and nuclear factor-xcexaB (Xanthoudakis et al., 1992).
Immunohistochemistry has been used to examine the subcellular distribution of APE in several different human tissues. The results show that levels vary significantly in different tissues (Duguid et al., 1995). APE expression in skin and intestine was tightly linked to cellular maturation. In most tissues, APE was detected primarily in the nucleus, where the APE staining pattern followed that of chromatin. In hepatocytes and some neurons, however, APE was detected primarily in the cytoplasm.
At this point in time, APE has not been associated with any particular pathologic conditions. Though clearly important to cellular function, specific diseases resulting from aberrations in this protein""s function are not known.
The present invention provides a method for identifying a premalignant or malignant condition in a human subject comprising determining the level of APE in cells from a sample from the human subject, wherein an elevated level of APE, as compared to the APE level in corresponding normal cells, indicates a premalignant or malignant condition in the human subject. As used herein the term xe2x80x9cnormal cellsxe2x80x9d means cells of the same tissue type, grown and at the same conditions and at the same cell cycle window and state of differentiation. In particular embodiments, the sample is selected from the group consisting of skin, muscle, facia, brain, prostate, breast, endometrium, lung, pancreas, small intestine, blood cells, liver, testes, ovaries, cervix, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow, kidney, peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, stool and urine.
In other embodiments, the premalignancy or malignancy is selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, cervix, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood tumor cells.
In a particular aspect the determining comprises evaluating APE protein levels. In some aspects, the determining may comprise evaluating APE transcript levels. In other aspects, the evaluating may comprise an immunoassay. In those aspects where APE transcript levels are evaluated the present invention may employ quantitative RT-PCR.
The present invention also provides an polyclonal antibody preparation which reacts immunologically with human APE. In other embodiments, there is provided a monoclonal antibody that reacts immunologically with human APE. The monoclonal antibody may further comprise a detectable label.
In other embodiments, the present invention provides a method for determining the premalignant or malignant state of a cell comprising determining the level of APE in the cell, wherein an elevated level of APE, as compared to the APE level in a corresponding normal cell, indicates a premalignant or malignant state in the cell.
In particular aspects, the determining comprises the steps of: disrupting the cell; contacting the disrupted cell with an antibody that reacts immunologically with APE; and quantitating the amount of APE bound to the antibody. In certain embodiments the cell may disrupted by detergent lysis, freeze-thaw, sonication, osmotic shock or manual rupture. In various independent embodiments the quantitating may be by ELISA or by RIA.
In other embodiments, the determining comprises the steps of: isolating mRNA from the cell; subjecting the mRNA to reverse transcription to produce cDNA; and quantitating APE cDNA by PCR.
The present invention contemplates a kit for identifying APE levels comprising: a first antibody that binds immunologically to APE; and an agent for detection of APE bound to the first antibody. In particular embodiments, the agent may comprise a second antibody or polyclonal sera that binds immunologically to an epitope of APE other than that bound by the first antibody. In other embodiments, the agent may be a second antibody that binds to the Fc region of the first antibody. In more particular aspects the second antibody comprises a detectable label.
The present invention further provides a method for diagnosis of premalignant or malignant condition in a human subject which comprises: administering to the subject an imaging agent comprising antibodies which react immunologically with APE bound to a label which is detectable by an external scan of the subject; and externally scanning the subject to determine whether there is a localized concentration of the imaging agent. In certain aspects the label may be a radioactive label or may be detectable by an X-ray, positron emission or magnetic resonance imaging scanning of the subject.
Also provided by the present invention is a method for therapeutic treatment of an APE-related premalignant or malignant condition in a human subject comprising administering to the patient an effective therapeutic amount of an agent that reduces the APE activity level in premalignant or malignant cells of the human subject. In particular embodiments the reducing comprises inhibiting expression of an APE gene in the cells. In other embodiments, the reducing comprises inhibiting APE function in the cells. In certain aspects the inhibiting may comprise contacting the cells with antisense APE expression constructs. Alternatively, the inhibiting comprises contacting the cells with antibodies that bind immunologically to APE.
It has now been determined that decreased amounts of APE are present in the cells undergoing and/or likely to undergo apoptosis. This discovery has enabled the use of APE as a marker for apoptosis, to which the present invention is generally addressed. Thus, in one embodiment, the invention provides methods and materials for the specific and sensitive assay of cells to assist in the identification of an apoptotic condition of the cells, and for modulating the apoptotic behavior of cells. The inventive methods and materials are expected to be highly useful in many fields including inter alia in the study and administration of cancer and cancer therapies.
Thus in alternative embodiments, the present invention provides a method for identifying apoptosis in a cell comprising (i) obtaining a sample and (ii) determining the level of APE in the sample, wherein a decreased level of APE, as compared to a normal APE level for a cell of the same type, indicates that the cell is undergoing apoptosis. In particular embodiments, the level is decreased by at least about 50% compared to the control. In other embodiments, the level is decreased by at least about 75% compared to the control. In yet other embodiments, the level is decreased by at least about 90% compared to the control.
In preferred embodiments, the cell is a tumor cell that has been subjected to chemotherapy, radiotherapy or gene therapy. In particular embodiments, the tumor cell is selected from the group consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, cervix, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood tumor cells.
In certain aspects of the present invention the determining comprises evaluating APE protein levels. The determining may comprise evaluating APE transcript levels. Alternatively, the evaluating may comprise an immunoassay or quantitative RT-PCR.
The present invention describes a method for monitoring the efficacy of a cancer therapy comprising (i) administering a therapeutic agent to cancer cells of a subject and (ii) determining the level of APE in a cancer cell from the subject, wherein a decreased level of APE, as compared to the APE level for the cells prior to the administering, indicates that the cell is undergoing apoptosis and the therapy is effective.
In alternative embodiments, the present invention provides a method for determining the apoptotic state of cells in a sample comprising (i) obtaining a sample and (ii) determining the level of APE in cells of the sample, wherein a decreased level of APE, as compared to the APE level in a cell of the same type, indicates that the cells are undergoing apoptosis. In this context, xe2x80x9ca cell of the same typexe2x80x9d means a treated tumor cell as compared to an untreated tumor cell, or a diseased cell as compared to a normal cell.
In yet another embodiment, there is provided a method for inducing apoptosis in a cell comprising reducing the amount of APE activity in the cell. In particular embodiments, the reducing comprises inhibiting expression of an APE gene in the cell. More particularly, the inhibiting comprises providing to the cell an APE antisense expression construct. In other embodiments, the reducing comprises inhibiting APE function. In certain embodiments the inhibiting may comprise providing to the cell an anti-APE single-chain antibody expression construct. In other embodiments, the inhibiting may comprise providing to the cell an inactive APE fragment, peptide or mimetic that competes with APE for binding to an APE substrate. To the extent that APE has an apoptotic activity an inactive fragment is defined as an APE fragment that does not have such an apoptotic function but retains all other APE-like functions.
The present invention, in an alternative embodiment, describes a method for inhibiting apoptosis in a cell comprising increasing APE activity in the cell. In particular, the increasing may comprise providing to the cell APE, or an active fragment thereof. In one embodiment, the providing comprises contacting the cell with an expression construct encoding APE or an active fragment thereof. In another embodiment, the providing may comprise contacting the cell with a purified APE polypeptide. In certain aspects the cell may be a T-cell infected with a human immunodeficiency virus.
In another inventive aspect, the present invention provides a method for enhancing the sensitivity of a tumor cell to a chemotherapy, a radiotherapy or gene therapy comprising reducing the amount of APE activity in the cell. The reducing may comprise inhibiting expression of an APE gene in the cell. Alternatively, the reducing may comprise inhibiting APE function.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.